The neoadjuvant paradigm reinvigorated: a review of pre-surgical immunotherapy in HNSCC.

BACKGROUND: There remains up to a 50% recurrence rate in advanced p16- head and neck squamous cell carcinoma with current standard of care treatment. In an attempt to improve survival, multiple trials administering induction or neoadjuvant chemotherapy have been conducted but none demonstrated improved overall survival. The established efficacy of immune checkpoint inhibitors in the recurrent and metastatic setting has produced widespread interest in their neoadjuvant use. PURPOSE: To survey the landscape of active neoadjuvant immunotherapy trials in head and neck squamous cell carcinoma and summarize and synthesize currently available outcomes from these trials. CONCLUSIONS: Neoadjuvant immunotherapy has proven safe and well tolerated in head and neck squamous cell carcinoma with encouraging efficacy results, including relatively high rates of pathologic response. Ongoing studies offer an opportunity to study immune responses in vivo. PD-L1 positivity, high tumor mutational burden and infiltration of NK cells, CD8, CD26 and Tim3 positive lymphocytes at time of surgery have been correlated with pathologic responses. We await updated reports of disease free survival and overall survival data and results of ongoing phase III studies utilizing neoadjuvant immunotherapy to determine if this treatment paradigm will have a place in the standard of care treatment in head and neck squamous cell carcinoma.

A Systematic Approach to Working With Medical Learners in Difficulty: A Faculty Development Workshop.

Introduction: For medical educators, applying a systematic approach to working with struggling learners (learners in difficulty) can improve faculty success and satisfaction with the remediation process. Use of the familiar SOAP diagnostic framework can ensure that faculty develop a thorough differential diagnosis and target their interventions to address underlying issues affecting learner success. Methods: We developed a workshop to teach medical education faculty essential skills for supporting learners in difficulty. Teaching methods included didactic presentation, large-group discussion, and small-group work with role-plays. Over three 2-hour sessions, participants learned to assess a learner in difficulty, develop an initial remediation plan, and evaluate their learning system with the goal of improving support to learners in difficulty. Evaluation included pre- and postsession assessment of learner self-perceived confidence and skill with working with struggling learners, as well as brief postsession evaluation. Results: Ninety-nine faculty participated in the Learners in Difficulty workshop over 7 years. Participants' overall rating of the workshop was 4.9 (1 = poor, 5 = outstanding). Pre- and postworkshop evaluation showed a statistically significant increase in perceived self-confidence to "Meet the needs of a struggling learner," from an average of 4.4 to 7.6 on a 10-point scale (mean Δ = 3.2; 95% confidence interval, 2.6-3.8; p < .001). Discussion: This workshop provides a stepwise approach to working with learners in difficulty and assessing participants' educational systems to identify strengths and weaknesses. Evaluations indicated participants felt more confident in their ability to engage in this topic following completion of the workshop.

14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides.

MOTIVATION: The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets and to prioritize the downstream analysis of >2000 potential interactors identified in high-throughput experiments. RESULTS: Here, a comprehensive set of 14-3-3-binding targets from the literature was used to develop 14-3-3-binding phosphosite predictors. Position-specific scoring matrix, support vector machines (SVM) and artificial neural network (ANN) classification methods were trained to discriminate experimentally determined 14-3-3-binding motifs from non-binding phosphopeptides. ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from -6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60. Blind prediction showed that all three methods outperform two popular 14-3-3-binding site predictors, Scansite and ELM. The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome. Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful. AVAILABILITY AND IMPLEMENTATION: A standalone prediction web server is available at http://www.compbio.dundee.ac.uk/1433pred. Human candidate 14-3-3-binding phosphosites were integrated in ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome database.

Bioinformatic and experimental survey of 14-3-3-binding sites.

More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.

IRAK1-independent pathways required for the interleukin-1-stimulated activation of the Tpl2 catalytic subunit and its dissociation from ABIN2.

The protein kinase Tpl2 (tumour progression locus 2) is activated by LPS (lipopolysaccharide), TNFalpha (tumour necrosis factor alpha) and IL (interleukin)-1. Activation of the native Tpl2 complex by these agonists requires the IKKbeta {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase beta}-catalysed phosphorylation of the p105/NF-kappaB1 subunit and is accompanied by the release of the catalytic subunit from both p105/NF-kappaB1 and another subunit ABIN2 (A20-binding inhibitor of NF-kappaB 2). In the present study we report that IL-1 activates the transfected Tpl2 catalytic subunit in an HEK (human embryonic kidney)-293 cell line that stably expresses the IL-1R (IL-1 receptor), but does not express the protein kinase IRAK1 (IL-1R-associated kinase). In these cells IL-1 does not activate IKKbeta or induce the phosphorylation of p105/NF-kappaB1, and nor does the IKKbeta inhibitor PS1145 prevent the IL-1-induced activation of transfected Tpl2. However, the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null cells or activation of the endogenous Tpl2 complex in IRAK1-expressing cells is suppressed by the protein kinase inhibitor PP2 by a mechanism that does not involve inhibition of Src family protein tyrosine kinases. The IL-1-stimulated activation of transfected Tpl2 is accompanied by its phosphorylation at Thr290 and Ser400 and by enhanced phosphorylation of Ser62, which we demonstrate are autophosphorylation events catalysed by Tpl2 itself. We further show that IL-1 triggers the dissociation of Tpl2 from co-transfected ABIN2 in IRAK1-null IL-1R cells, which is not suppressed by PP2 or by the inhibition of Tpl2 or IKKbeta. These studies identify two new signalling events involved in activation of the native Tpl2 complex by IL-1. First, the IRAK1-, IKKbeta- and PP2-independent dissociation of Tpl2 from ABIN2; secondly, the IRAK1- and IKKbeta-independent, but PP2-sensitive, activation of the Tpl2 catalytic subunit.

Interleukin-1 (IL-1) induces the Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitate NEMO binding and the activation of IkappaBalpha kinase.

Interleukin 1 (IL-1) has been reported to stimulate the polyubiquitination and disappearance of IL-1 receptor-associated kinase 1 (IRAK1) within minutes. It has been thought that the polyubiquitin chains attached to IRAK1 are linked via Lys48 of ubiquitin, leading to its destruction by the proteasome and explaining the rapid IL-1-induced disappearance of IRAK1. In this paper, we demonstrate that IL-1 stimulates the formation of K63-pUb-IRAK1 and not K48-pUb-IRAK1 and that the IL-1-induced disappearance of IRAK1 is not blocked by inhibition of the proteasome. We also show that IL-1 triggers the interaction of K63-pUb-IRAK1 with NEMO, a regulatory subunit of the IkappaBalpha kinase (IKK) complex, but not with the NEMO[D311N] mutant that cannot bind K63-pUb chains. Moreover, unlike wild-type NEMO, the NEMO[D311N] mutant was unable to restore IL-1-stimulated NF-kappaB-dependent gene transcription to NEMO-deficient cells. Our data suggest a model in which the recruitment of the NEMO-IKK complex to K63-pUb-IRAK1 and the recruitment of the TAK1 complex to TRAF6 facilitate the TAK1-catalyzed activation of IKK by the TRAF6-IRAK1 complex.

Interleukin-1 stimulated activation of the COT catalytic subunit through the phosphorylation of Thr290 and Ser62.

The protein kinase COT/Tpl2 is activated by interleukin-1 (IL-1), TNFalpha and lipopolysaccharide, and its activation by these agonists involves the IkappaB kinase beta (IKKbeta) catalysed phosphorylation of the p105 regulatory subunit. Here, we show that COT activation also requires catalytic subunit phosphorylation, since IL-1beta induced a 5-10-fold activation of a COT mutant unable to bind p105. Activation was paralleled by the phosphorylation of Thr290 and Ser62 and unaffected by the IKKbeta inhibitor PS1145 at concentrations which prevented the degradation of IkappaBalpha. Mutagenesis experiments indicated that COT activation is initiated by Thr290 phosphorylation catalysed by an IL-1-stimulated protein kinase distinct from IKKbeta, while Ser62 phosphorylation is an autophosphorylation event required for maximal activation.

Characterization of the reversible phosphorylation and activation of ERK8.

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15-20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.

PKD: a new protein kinase C-dependent pathway in platelets.

Protein kinase D (PKD, also known as PKCmu) is closely related to the protein kinase C superfamily but is differentially regulated and has a distinct catalytic domain that shares homology with Ca(2+)-dependent protein kinases. PKD is highly expressed in hematopoietic cells and undergoes rapid and sustained activation upon stimulation of immune receptors. PKD is regulated through phosphorylation by protein kinase C (PKC). In the present study, we show that PKD is expressed in human platelets and that it is rapidly activated by receptors coupled to heterotrimeric G-proteins or tyrosine kinases. Activation of PKD is mediated downstream of PKC. Strong agonists such as convulxin, which acts on GPVI, and thrombin cause sustained activation of PKC and PKD, whereas the thromboxane mimetic U46619 gives rise to transient activation of PKC and PKD. Activation of PKD by submaximal concentrations of phospholipase C-coupled receptor agonists is potentiated by G(i)-coupled receptors (eg, adenosine diphosphate and epinephrine). This study shows that PKD is rapidly activated by a wide variety of platelet agonists through a PKC-dependent pathway. Activation of PKD enables phosphorylation of a distinct set of substrates to those targeted by PKC in platelets.